gamma-Secretase is the proteolytic activity that catalyzes the final step in the generation of the amyloid beta-peptide (AP) from the transmembrane Alzheimer amyloid precursor protein (APP). Growing evidence suggests that the presenilin proteins (PS1 and PS2) are required in order for gamma-secretase cleavage to occur. The PS proteins are endoproteolytically cleaved, generating an N-terminal fragment (NTF) and a C-terminal fragment (CTF). The NTF and CTF remain stably associated as heterodimers and assemble together with the partner proteins known as nicastrin, aph-1, and pen-2 proteins in order to form the high molecular weight (approximately 10/6 kDa) complex that is believed to constitute the functional gamma secretase. Gamma-Secretase cleavage is unusual in that it apparently occurs within the anhydrous environment of the membrane bilayer. The realization that numerous transmembrane proteins are processed in a similar fashion has inspired the formulation of a general concept: regulated intramembrane proteolysis, or RIP. The general goal of this proposal is to advance our understanding of the structure and function of PS proteins. In Aim 1 mutant PS molecules will be designed that permit the localization of domains that are important for (i) endoproteolytic processing of PS, and for (ii) intramembranous processing of APP to liberate Abeta40, Abeta42, and the APP intracellular domain (AICD). Successive truncation and point mutagenesis strategies will be utilized. In Aim 2, we will elucidate the conditions required for generating aspartyl proteinase activity from recombinant PS1 and its partners nicastrin, aph-1 and pen-2, following their expression in insect cells. This investigation will permit the identification of the minimal functional unit for gamma-secretase activity and potentially support the notion that PS1 is indeed the catalytic component of the gamma-secretase complex.